The B Cell Biology Scientific Research Support Component (SRSC) will assist in the generation of novel vaccines that target broad neutralizing antibodies (BnAbs) or other protective B-cell precursors by B lineage immunogen design. The novel vaccines generated by the CHAVI-ID B Cell Focus will be designed to activate the unmutated ancestors (UA) of highly mutated BnAb B cells isolated by flow cytometry. As a parallel strategy, this B Cell Biology SRSC will first isolate human B-cell precursors, and drives their maturation in vitro. BnAb precursors will then be identified and allowed to proliferate, mature and undergo Ig class-switching (CSR) in vitro. In this manner, the B Cell Biology SRSC will complement the work of the B Lineage Immunogen Design teams by identifying and selecting BnAb precursors -unmutated ancestors B cells - that are comparable to, and possibly better than, those inferred from BnAb clonal lineages. This support component will identify, isolate, characterize, and activate de novo B cells capable of generating BnAb responses, allowing the direct study of BnAb UA and intermediate antibody (IA) B cells. Specific Aims Aim 1. Culture Env-specific immature B cells from human bone marrow in vitro and drive them to express AID and undergo Ig class-switch recombination in vitro to characterize BnAb maturation pathways. Aim 2. Culture Env-specific immature B cells from humanized Velocimmune mice in vitro and drive antigen-specific B cells to compete and mature by AID expression and Ig CSR to characterize BnAb maturation pathways. Aim 3. Immunize immunoglobulin humanized mice with candidate Env vaccines to determine their immunogenicity by characterizing serum antibody responses and hybridoma lines.